Method and device for high throughput cell deformability measurements

ABSTRACT

A system is disclosed that enables the automated measurement of cellular mechanical parameters at high throughputs. The microfluidic device uses intersecting flows to create an extensional flow region where the cells undergo controlled stretching. Cells are focused into streamlines prior to entering the extensional flow region. In the extensional region, each cell&#39;s deformation is measured with an imaging device. Automated image analysis extracts a range of independent biomechanical parameters from the images. These may include cell size, deformability, and circularity. The single cell data that is obtained may then be used to in a variety of ways. Scatter density plots of deformability and circularity may be developed and displayed for the user. Mechanical parameters such as deformability and circularity may be gated or thresholded to identify certain cells of interest or sub-populations of interest. Similarly, the mechanical data obtained using the device may be used as cell signatures.

RELATED APPLICATION

This Application claims priority to U.S. Provisional Patent ApplicationNo. 61/385,268 filed on Sep. 22, 2010. Priority is claimed pursuant to35 U.S.C. § 119. The above-noted patent application is incorporated byreference as if set forth fully herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant No.W81XWH-10-1-0519, awarded by the U.S. Army, Medical Research andMateriel Command and Grant No. 0930501, awarded by the National ScienceFoundation. The Government has certain rights in this invention.

FIELD OF THE INVENTION

The field of the invention generally relates to microfluidic devices andmethods for measuring the size, circularity, and deformability of cells.The field of the invention also pertains to devices and methods forutilizing these measured parameters as biomarkers and cell phenotypeidentification purposes.

BACKGROUND

There is growing evidence that cell deformability (i.e., the degree towhich a cell changes shape under an applied load) is a useful indicatorof abnormal cytoskeletal changes and may provide a label-free biomarkerfor determining cell states or properties such as metastatic potential,cell cycle stage, degree of differentiation, and leukocyte activation.Clinically, a measure of metastatic potential could guide treatmentdecisions, or a measure of degree of differentiation could preventtransplantation of undifferentiated, tumorigenic stem cells inregenerative therapies. For drug discovery and personalized medicine, asimple measure of cytoskeletal integrity could allow screening forcytoskeletal-acting drugs or evaluation of cytoskeletal drug resistancein biopsied samples. Currently, these applications often require costlydyes, antibodies, and other reagents, along with skilled technicians toprepare samples. A simple label-free deformability measurement in whichcells are minimally handled has the potential to greatly reduce costsand allow routine cell screening and classification in clinical andresearch applications.

Current platforms and techniques that measure cell deformability havesuffered from a number of limitations. These include low throughput aswell as inconsistent results. As a result, these technologies have nothad any significant clinical impact. A wide variety of platforms havebeen engineered to perform mechanical measurements on cells. Generally,these techniques can be divided into two categories based on the samplesthey act on: bulk and single-cell. Bulk platforms, such asmicrofiltration, tend to have high throughput, but they yield oneendpoint measurement and do not take into account heterogeneity withinthe sample population of cells. Disease may develop from abnormalitiesin a single cell thus accurately detecting rare events or localvariations is important and bulk measurement of these types of samplesmay result in misleading averages. Single-cell platforms that can assaythis heterogeneity include micropipette aspiration, atomic forcemicroscopy (AFM), magnetic bead-based rheology, microfluidic opticalstretching, and biophysical flow cytometry.

However, these approaches are typically optimized for biophysicsresearch and operate at low rates at around 1 cell/minute for AFM andoptical stretching. Applications in clinical diagnostics or drugscreening will necessarily require large sample sizes to obtainstatistically significant results. This cannot reasonably be achievedusing low throughputs on the order of 1 cell/minute. Further, thesetechniques also suffer from other disadvantages. AFM, for example,requires a skilled operator and measurements are slow. Rheologicaltechniques can yield drastically different mechanical properties thatare difficult to standardize even amongst a single cell type. Inaddition, these techniques require microscopic observation at highmagnification for a period of time such that the overall throughput isvery low (<<1 cell/minute). The manual, low-throughput nature of currentmethods that measure cell mechanical properties has limited thecapability for development of practical biomechanical biomarkers fortranslational use, as well as limited the progress of understandingmolecular components underlying cell mechanical properties.

SUMMARY

In one embodiment, a microfluidic device is disclosed that enables theautomated measurement of cellular mechanical parameters at highthroughputs greater than 1,000 cells/second. The microfluidic deviceuses intersecting flows to create an extensional flow region where thecells undergo controlled stretching. Cells are focused into streamlines(e.g., a continuous stream of single cells in a streamline) prior toentering the extensional flow region. In the extensional region, eachcell's deformation is measured with an imaging device. Automated imageanalysis extracts a range of independent biomechanical parameters fromthe images. These may include cell size, deformability, and circularity.The single cell data that is obtained may then be used in a variety ofways. For example, scatter density plots of deformability andcircularity may be developed and displayed for the user similar to theway in which traditional flow cytometry scatter plots are used.Mechanical parameters such as deformability and circularity may be gatedor thresholded to identify certain cells of interest or sub-populationsof interest. Similarly, the mechanical data obtained using the devicemay be used as cell signatures.

Generally, the method for high throughput cell deformabilitymeasurements involves positioning the cells along a focused path orstreamline at relatively high flow rates. The cells are then deliveredto an extensional region wherein each cell is subject to uniformlycontrolled deformation (e.g., cell stretching). The imaging devicecaptures this controlled deformation whereby the images are subject tomorphological analysis to determine cell size, cell deformability, andcell circularity. This data can then be quickly presented to the user ina useful format (e.g., scatter plot) or further processed to present theuser with useful information regarding the tested cells. For example,the method may be used to screen a sample of cells for a diseased state(e.g., cancer), identify useful information regarding stem celldifferentiation, or be further subject to additional data mining forpredictive information. The method may also complement existing cellularanalysis tools to provide more confidence in decision making. The methodis also beneficial in that it reduces costs because of the reducedreagent consumption. Similarly, there is a reduction in labor costsbecause the automatic method eliminates time consuming steps such aspipetting, centrifugation, etc.

In one embodiment of the invention, a system for measuring particle(e.g., cell) deformability includes a substrate containing first andsecond microfluidic channels dimensioned to carry cells therein and anextensional region comprising an intersection of the first and secondmicrofluidic channels, wherein the first and second microfluidicchannels intersect in substantially opposite directions. The systemincludes at least one outlet channel coupled to the extensional regionand an imaging device configured to capture a plurality of image framesof cells passing through the extensional region and at least oneprocessor configured to calculate a morphological parameter of the cell.These parameters may include cell size, cell deformability, and cellcircularity of cells passing through the extensional region. Additionalparameters include cell shape, cell granularity, and intracellularstructure.

In another embodiment of the invention, a method of measuring particle(e.g., cell) deformability includes focusing a plurality of cells infirst and/or second microfluidic channels dimensioned to carry cellstherein. For example, in one configuration, cells are carried inopposing microfluidic channels that intersect as described below. Inanother configuration, cells are only carried by one of the twointersecting microfluidic channels. The cells of the first and/or secondmicrofluidic channels are then intersected in an extensional regionconfigured to apply stress to cells passing therein. A plurality ofimage frames of the cells are obtained, wherein the plurality of imageframes contain images of cells prior to entering the extensional regionand during exposure to the extensional region. One or more dimensionalparameters of the cells are measured from the plurality of image framesprior to entering the extensional region and during exposure to theextensional region. The deformability of a cell is determined based atleast in part on the change of the one or more dimensional parametersoccurring during exposure to the extensional region.

In another embodiment, a system for measuring particle deformabilityincludes a substrate containing first and second microfluidic channelsdimensioned to carry cells therein. The system includes an extensionalregion comprising an intersection of the first and second microfluidicchannels, wherein the first and second microfluidic channels intersectin substantially opposite directions. At least one outlet channel iscoupled to the extensional region. The system further includes anoptical collector configured to capture diffracted or refracted lightfrom cells passing through the extensional region and at least oneprocessor configured to calculate a morphological parameter of theparticle passing through the extensional region.

In another embodiment, a system for measuring particle deformabilityincludes a substrate containing a microfluidic channel dimensioned tocarry cells therein and an extensional region comprising a junctionwherein the velocity of the flow in the incoming flow direction abruptlydecreases to substantially zero. At least one outlet channel is coupledto the junction of the extensional region. The system includes animaging device configured to capture a plurality of image frames ofcells passing through the extensional region and at least one processorconfigured to calculate a morphological parameter of the particlepassing through the extensional region.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a schematic view of a system for measuring celldeformability according to one embodiment.

FIG. 2 is a magnified view of region B of FIG. 1 illustrating cellsentering the extensional region.

FIG. 3 illustrates photographic images of a cell passing into anextensional region at various points of time.

FIG. 4 illustrates an automated cell tracking analysis andpost-processing algorithm according to one embodiment.

FIG. 5 illustrates a graph of the (0, r) mapping of a single cell.

FIG. 6 illustrates images of a single cell along with dimensionalindications for initial diameter (d), the maximum diameter (a) in thevertical direction, the minimum diameter (b) in the horizontaldirection, the area A of the observed cell, and the perimeter L of theobserved cell.

FIG. 7A illustrates scatter plots of cell deformability as function ofinitial diameter for human embryonic stem cells (hESCs) and hESCsundergoing differentiation (day 9).

FIG. 7B illustrates scatter plots of cell circularity as function ofinitial diameter for human embryonic stem cells (hESCs) and hESCsundergoing differentiation (day 9).

FIG. 8A illustrates scatter plots of cell deformability as function ofinitial diameter for undifferentiated mESC, differentiated stem cellswith Ad. Diff., and differentiated stem cells with EB Diff.

FIG. 8B illustrates scatter plots of cell circularity as function ofinitial diameter for undifferentiated mESC, differentiated stem cellswith adherent differentiation, and differentiated stem cells withembryoid body differentiation.

FIG. 9A illustrates scatter plots of cell deformability as function ofinitial diameter for normal cells (MCF10A), cancerous cells (MCF7), andthe same cancerous cell line modified to have increased motility ormetastatic potential (modMCF7).

FIG. 9B illustrates scatter plots of cell circularity as function ofinitial diameter for normal cells (MCF10A), cancerous cells (MCF7), andthe same cancerous cell line modified to have increased motility ormetastatic potential (modMCF7).

FIG. 10A illustrates scatter plots of cell deformability as function ofinitial diameter for HeLa cells (control), HeLa cells treated withLatrunculin A, and HeLa cells treated with Nocodazole.

FIG. 10B illustrates scatter plots of cell circularity as function ofinitial diameter for HeLa cells (control), HeLa cells treated withLatrunculin A, and HeLa cells treated with Nocodazole.

FIG. 11A illustrates one embodiment of a gating strategy that looks forcell percentage that exists in a region of high initial diameter andhigh deformability (darkened region in upper right quadrant).

FIG. 11B illustrates a scatter plot of cell deformability as function ofinitial diameter for a standard negative profile (non-cancerous).

FIG. 11C illustrates a scatter plot of cell deformability as function ofinitial diameter for a standard adenocarcinoma profile.

FIG. 12A illustrates a schematic view of a system for measuring celldeformability according to another embodiment.

FIG. 12B illustrates photographic images of a cell passing into anextensional region at various points of time.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

FIG. 1 illustrates a system 10 for measuring particle deformabilityaccording to one embodiment. Particles may include cells as well asother structures such as hydrogel particles, oil droplets, and the like.The system 10 has particular applicability for measuring deformabilityof cells but the system may also be used for measuring deformation ofother small particles. The system 10 is preferably implemented inmicrofluidic format on a substrate 12. The substrate 12 may include anynumber of structures commonly used for microfluidic applicationsincluding glass, polymers, or composite structures. For example, themicrofluidic features such as the inlets, outlets, extensional regions,channels, focusing regions, and the like may be formed in PDMS which isthen bonded to another substrate like glass. The microfluidic featuresillustrated in FIG. 1 may be designed using conventional software (e.g.,AutoCAD software available from Autodesk, San Rafael, Calif.).Transparency photomasks for the designs can be printed at 20,000 dotsper inch (CAD/Art Services, Inc., Bandon, Oreg., USA). Molds for replicamolding using these masks are prepared using negative photoresist, suchas SU-8 50 (MicroChem, Newton, Mass.). The negative photoresist is spunon a 4 inch Silicon wafer at 4000 rotations per minute. The coated waferis then soft baked at 65° C. for 5 minutes then 95° C. for 15 minutes.The wafer was then exposed under near UV radiation at 8.0 mW/cm2 for 30seconds. A post-exposure bake of the wafer is carried out at 65° C. for2 minutes then at 95° C. for 3.5 minutes. The unexposed photoresist isdeveloped in SU-8 Developer (MicroChem) until an isopropyl alcohol rinseproduced no white film. The height of the microfluidic features may varybut is generally on the order of several or more microns (e.g., about 28μm). Of course, the height used in a particular system 10 may vary.

The mold that is created can be taped to the lower plate of a Petri dishwith features facing up and an approximately 6 mm layer of Sylgard 184Silicone Elastomer (Dow Corning, Midland, Mich., USA),polydimethylsiloxane (PDMS), mixed 10 parts base to 1 part curing agent,is poured on top. The cast mold was placed in a vacuum chamber and thechamber was evacuated for 30 minutes to remove air from the curingpolymer. It was then moved to an oven set to 65° C. for 3 hours. Thedevices were cut from the mold and inlet and outlets were punched intothe cured PDMS polymer. The devices were then placed in a plasma cleaneralong with slide glasses to be activated. After a 30 second exposure toair plasma the activated surfaces of PDMS and glass were placed incontact to form permanent covalent bonds between the two materials.

Still referring to FIG. 1, the system includes an inlet 14 through whicha fluid laden with cells is flowed. Located in or adjacent to the inlet14 is an optional filter 16 that may be used to filter out dust oraggregate particulate matter. The filter 16 may include, for instance,posts or others physical impediments to flow of particulate matter abovea certain size. Of course, cells and other particles of interest canflow past the filter 16. A pump 18 is connected to the inlet 14 via aconduit 20 or the like. The pump 18 may include a syringe pump (e.g.,PHD 2000 syringe pump, Harvard Apparatus, Holliston, Mass.), pressurepump, or other pumping device known to those skilled in the art. Thepump 18 pumps a solution containing cells into the inlet 14 at a uniformflow rate. The actual rate of flow may be adjusted or tuned by the pump19 to achieve desired mechanical deformations of the cells. Forinstance, low flow rates may result in non-uniform mechanical stretchingwhile high flow rates may result in cells that are stretched beyond theimaging window (described in more detail below) which results insaturation of the measurements. The optimal flow rate is one where thecells reach the center of the extensional flow (e.g., intersectingflows) where they deform in a non-saturating amount. Flow rates varydepending on the size of the microfluidic features formed in the system10. For instance, channels having a diameter of 67 μm had an optimizedflow rate of 1075 μL/minute while a smaller diameter channel (44 μm) hadan optimized flow rate of 600 μL/minute. Cells are suspended in acarrier fluid that is run through the system 10. The density of cellsmay vary but generally falls within the range of about 200,000 cells/mLand about 500,000 cells/mL.

Downstream of the inlet 14 the system 10 includes two branch channels22, 24 operatively coupled to the common inlet 14. Flow thus occurs inthe direction of arrows A of FIG. 1. The branch channels 22, 24 bothlead to respective focusing regions 26, 28. The focusing regions serveto align the cells to the same streamlines prior to entering theextensional region 40 whereby cells undergo deformation. This ensuresthat each cell, traveling at the same downstream velocity, experiencesthe same force field upon entering the extensional region 40. A varietyof different focusing techniques can be used including hydrodynamicfocusing, sheath focusing, dielectrophoretic focusing, ultrasonicfocusing and inertial focusing. Inertial focusing has the advantage thatpinching sheath flows are not needed to steer cells to a preciseposition, an improvement that leads to a single input robust technology.Inertial focusing using curving, confined flows is illustrated inFIG. 1. Details regarding this type of inertial focusing may be seen in,for example, D. R. Gossett et al., “Particle focusing mechanisms incurving confined flows,” Analytical Chemistry, 81, 8459 (2009), which isincorporated by reference herein.

Still referring to FIG. 1, the cells leave the respective focusingregions 26, 28 with the cells aligned along streamlines contained infirst and second microfluidic channels 30, 32. The cells proceed alongthe first and second microfluidic channels 30, 32 whereby theextensional region 40 is reached. The extensional region 40 includes anintersection of the first and second microfluidic channels 30, 32 insubstantially in opposite directions. That is to say, the first andsecond microfluidic channels 30, 32 are substantially coaxially alignedwith respect to one another. Namely, the flow and cells containedtherein are directed head-on at one another in a crossing channelforming a + pattern to create a region of large deceleration whereby thecells undergo mechanical stress thereby causing, depending on the natureof the cell, deformation. The system 10 of FIG. 1 further includes twomicrofluidic outlet channels 34, 36 that are oriented substantiallyperpendicular with respect to first and second microfluidic channels 30,32. The microfluidic outlet channels 34, 36 terminate at outlets 38. Theoutlets 38 may be coupled to tubing (not shown) with free ends directedto a waste receptacle or the like.

FIG. 2 is enlarged view of region B of FIG. 1 illustrating the first andsecond microfluidic channels 30, 32 as well as the first and secondmicrofluidic outlet channels 34, 36 intersecting in the extensionalregion 40. As explained below, region B may coincide substantially withthe field of view (FOV) of the imaging system. Cells 50 are illustratedaligned along a lateral equilibrium streamline (X_(eq)) prior toentering the extensional region 40. The extensional region 40 containspurely stretching flow. As the cells 50 sequentially enter theextensional region 40, they are subject to the stretching flow withinthe extensional region 40 and undergo deformation. As explained below,this deformation is then imaged whereby one or more dimensionalparameters are extracted. These dimensional parameters are then used todetermine cell size, cell deformability, and cell circularity, or othermorphological parameters such as cell shape, cell granularity, andintracellular structure. Cell shape is generally a function that definesthe edge of the object. The shape could be spherical, ellipsoid, starshaped, etc. Granularity refers to the spatial frequency of variation inthe optical contrast/index of refraction within a cell. Intercellularstructure refers to the size and shape of objects internal to the cellthat have optical contrast. This may include cellular organelles such asthe cell nucleus or the like. Moreover, it may be desirable to measureone or more morphological parameters prior to the cells 50 entering theextensional region 50

The system 10 operates with a high throughput. Preferably, thousands ofcells 50 individually flow at over 1 meter/second into the extensionalregion 40. After the cells 50 have been subject to the stretchingforces, the cells 50 leave region B via one of microfluidic outletchannels 34, 36. As the cells 50 leave region B, another cell 50 canenter the extensional region 40 whereby it is subject to substantiallythe same deformation forces. This next cell 50 is imaged and leavesregion B. This process is repeated many times over to enable theprocessing of over 1,000 cells/second in series. For example, nearly2,000 deformations per second may be reached using the current system 10which is more than three (3) orders of magnitude over currentstate-of-the-art methods for measuring the mechanical properties ofcells.

As an alternative to the system 10 of FIG. 1, one of the first andsecond microfluidic channels 30, 32 may be omitted to form a junction inthe manner of a T-shape as shown in FIG. 12A. For example, the Tstructure may include a microfluidic channel 30 (inlet) and two outletchannels 34, 36. Thus, in this alternative embodiment fluid flows towardthe junction whereupon the velocity of the flow in the incoming flowdirection abruptly decreases to substantially zero. In this alternativeembodiment, the other aspects of FIG. 1 remain the same (e.g., focusing,imaging, etc.). FIG. 12B illustrates a series of images taken of a cell50 passing through a device of the type illustrated in FIG. 12A.

Referring back to FIG. 1, the system 10 includes an imaging system 60that enables high speed images to be taken of cells 50 passing within afield of view (FOV). The FOV includes the extensional region 40 as wellas some portion of the upstream segments of first and secondmicrofluidic channels 30, 32. Similarly, the FOV may also includeportions of the first and second microfluidic outlet channels 34, 36.For instance, as seen in FIG. 1, the FOV may be substantiallycoextensive with region B. The imaging system 60 may include amagnifying objective lens 62 that is used to magnify the view. Forexample, the magnifying objective 62 may include a 10× objective (NikonJapan 10×/NA 0.30) that is affixed to a Nikon Eclipse Ti invertedmicroscope (not shown). The imaging system 60 further includes animaging device 64 capable of obtaining a plurality of image frames of aFOV. The imaging device 64, in one aspect, is a digital high speedcamera capable of capturing several thousand frames per second. Forexample, the imaging device 64 preferably operates at more than 50,000records per second and an exposure time per record of ≤2 microseconds.While digital imagery may be obtained with a high-speed camera, otherimaging modalities capable of capture rates described above may be used.For example, in some alternative embodiments, records of the FOV may berecorded by devices not having an array of pixels to capture an imageframe. In this regard, data is collected by an optical collector thatwould replace the imaging device 64. This may include light collectedonto a photodiode or photomultiplier tube (PMT). These devices mayrecord, for example, optical contrast or scattered light which may beused to derive one or more morphological parameters.

As one example, the imaging device 64 may include a digital high-speedvideo camera, Phantom v7.3 (Vision Research, Inc., Wayne, N.J., USA),connected to the microscope via a c-mount for image capture. Camerasettings can be controlled with Phantom Camera Control (Vision Research,Inc.). The frame rate of the camera is limited by the chosen pixelresolution. For the larger diameter (67 μm) device, 256×32 pixels wasused while 128×24 pixels was used for the smaller diameter (44 μm)device. The resulting frame rates were 133,333 per second and 173,913per second, respectively. The minimum allowable exposure time, 1 μs, wasused for both devices. The device was aligned at the center of the FOV.The aperture to the imaging device 64 was half-closed to focus light andreduce scatter. Light intensity was adjusted to maximize the contrastbetween the cell walls and the exterior fluid.

FIG. 3 illustrates images of a cell 50 passing into an extensionalregion at various time periods. The cell 50 is a MCF7 cell and is imagedat 0, 7, 14, 21, and 28 μs. Stretching of the cell 50 is clearly seen inthe 28 μs image.

The system 10 further includes a computer 70 containing at least oneprocessor therein 72. The computer 70 is used for data analysis ofindividual image frames obtained from the imaging device 64. Thecomputer 70 may also be used for data acquisition purposes to storeeither permanently or temporarily image frames or other representativedata. The computer 70 may also be used for post-processing analysis suchas modeling, classification/regression tree analysis (e.g., trainingsets for classification trees (CART)). In still other aspects, thecomputer 70 may be integrated with other aspects of the system 10. Forinstance, the computer 70 may be used to control the flow rate of thepump 18. The computer 70 and processor(s) 72 contained therein are usedto execute software contained therein for image analysis. The computer70 also includes a display 74 that may be used to display one or moredimensional parameters of the cells 50 passing through the extensionalregion 40. For example, the display 74 may display to the user acytometry-like scatter plot of data such as deformability or circularityas a function of initial cell diameter for a large batch of cells.

The software used for image analysis may reside on or otherwise bestored in the computer 70 on a computer readable medium. Alternatively,the software used for image analysis may reside in a computer readablemedium at a remote location and executed with processor (not shown) thatitself is remote or local. In this alternative, the remote processor isaccessible via a network such as a wide area network (e.g., Internet) orlocal area network via the computer. In either instance, the softwarecontains script or instructions for the automated image analysis ofcells 50 passing through the system 10.

FIG. 4 illustrates an exemplary script or algorithm that is usable withthe system 10. The software includes functionality for the automatictracking and measurement of cells 50 passing through the device. Thesoftware also includes functionality for post-processing the raw datameasurements made from the individual frames. The software may beimplemented on any number of platforms known to those skilled in the artsuch as Matlab, etc. Starting at operation 100, a FOV from the imagingdevice 64 is subject to optional filtering to enhance cellidentification by adjusting contrast, gamma, and brightness. Apre-junction channel (pre-extensional region 40) is monitored asillustrated in operation 150 for the presence or absence of a cell 50.This pre-junction channel is located in first microfluidic channel 30upstream of the extensional region 40. If the cell 50 is not present,the next frame in the image sequence is looked at until a cell 50 isidentified in the pre-junction. Once the cell 50 is identified in theFOV, as seen in operation 200, The FOV is cropped and resized 10X toincrease the accuracy of measurements. Next, as seen in operation 250, acentering algorithm identifies the center of the cell 50 based on thecell position, shape, and local intensities. The image data at thispoint is represented in Cartesian coordinates as seen in operation 300.A polar coordinate representation of this image is then extracted(operation 350) whereby the cell as a function of (0, r). The cell wallsare found by examining changes in the intensity derivatives, anddiameters extracted every several degrees. The tracking algorithm thenmoves the FOV as seen in operation 370 to obtain additional frames ofthe cell 50 until it leaves the deformation-inducing extensional region40. Once the cell 50 has left, the process starts again at operation 100until a next cell 50 passes into view. At this point in the process, thecell 50 has been automatically tracked through the extension region 40and data 80 corresponding to cell diameters at intervals of severaldegrees (e.g., 2-3°).

Still referring to FIG. 4, post-processing (operation 450) is conductedon the data 80 to determine cell size, cell deformation, and cellcircularity. The cells 50 passing through the system 10 may either be ofa known phenotype or an unknown phenotype as illustrated by operation400. As part of the post processing operation 450, the size of the cell50 prior to entering the extensional region 40 is determined. The celldiameter (d) is determined as the average of the minimum cell diametersat 90°±30° to horizontal prior to entering the extensional region 40(approximately four (4) frames prior to entering extension region 40).By taking the size measurements from the vertical axis, noise due toblur attributed to high velocities in the horizontal direction isreduced. As the cell 50 enters the extension region 40 and changestrajectory the deformability and circularity parameters are measured. Inthe frame where the largest deformation is calculated, the correspondingcircularity is recorded as well. The deformability parameter isdetermined by the ratio a/b of the maximum diameter (a) in the verticaldirection at 90°±30° to the minimum diameter (b) in the horizontaldirection at 0°±30°. The circularity parameter is calculated by (4πA/L²)wherein A is the area of the observed cell and L is the perimeter of thecell.

FIG. 5 illustrates a graph of the (θ, r) mapping of a single cell 50.The minimum intensity in the plot is used as the cell edge and isrepresented in FIG. 5 by white line 52. The maximum diameter iscalculated by summing a₁ and a₂ (a=a₁+a₂) as illustrated in FIG. 5. Theminimum diameter is calculated by summing b₁ and b₂ (b=b₁+b₂) asillustrated in FIG. 5. Still referring to FIG. 5, the area A of theobserved cell is equal to the area under the white line 52 while theperimeter L of the cell is equal to the length of the white line 42.This information can be extracted from the data 80.

In one aspect of the invention, measurements of cells 50 whose initialdiameters measure greater than a maximum threshold value are discardedas these cells 50 are bigger than the smallest channel dimension and aredeforming to fit through the channels 30, 32. Measurements of cells 50whose initial diameters measure less than a minimum threshold value arealso discarded as confidence of these measurements is diminished by thesmall number of pixels per cell 50 at this size. For example, themaximum threshold may be set at 28 μm while the minimum threshold may beset to 5 μm. It should be understood that different threshold valuesthan those specifically set forth above may be used. In another aspect,measurements of cells 50 are discarded when their initial diameters aregreater than the third quartile plus 1.5× the interquartile range orless than the first quartile minus 1.5× the interquartile range.

FIG. 6 illustrates micrograph images of cells 50 showing the initialdiameter (d), the maximum diameter (a) in the vertical direction, theminimum diameter (b) in the horizontal direction, the area A of theobserved cell, and the perimeter L of the observed cell.

Referring back to FIG. 4, after collection of the data 80, the data 80undergoes post-processing in operation 450 to determine the initial size(d), deformability (a/b), and circularity (4πA/L²) of the cells 50 inthe manner described above. In one optional aspect of the invention asillustrated by operation 500, modeling software contained in thecomputer 70 (e.g., processor(s) 72) to obtain additional information onthe cells 50 like membrane elasticity or viscosity. For example, thetime dynamics of deformation and relaxation of the cell 50 after stressis removed can provide viscoelastic properties of the cells. A timeconstant for deformation may be extracted from a plot of the deformedshape of the cell 50 as a function of time. The same can be done forrelaxation of the cell 50 after removal of the stress.

In another alternative embodiment, operation 500 is omitted andtwo-dimensional (2D) scatter plots are generated of one or more of theparameters (e.g., size, deformability, circularity) as illustrated inoperation 550. The scatter plots may include, for example, initial sizeas a function of deformability or initial size as a function ofcircularity. The scatter plots may be displayed to the user on a display74 that is connected to the computer 70. The scatter plots contain largedatasets that allow the presentation of statistically rich deformabilitydata to the user, allowing definitive conclusions concerning cellmechanical properties. Moreover, users will be able to easily use thisinformation in part because of their familiarity to existing scatterplots for flow cytometry.

Instead of being displayed to the user in scatter plot format, thedatasets may be then tested in classification and regression treeanalysis as is illustrated in operation 600. This can be used to eitherdetermine cell phenotype 620 or it may be used to provide adeformability biomarker 640 that can then be used for identification androutine screening for clinical use.

Experiment No. 1—Differentiation of Embryonic Stem Cells

Experiments were conducted using the system to determine the ability tousing deformability as biomarker of the “stemness” of embryonic stemcells. Embryonic stem cells are known to have more deformable nuclei andare known to be more deformable than differentiated cells. In thisexperiment, the HSF-1line of human embryonic stem cells (hESCs) (46XYKaryotype) were tested in the system at flow rates between 700-800μL/min. FIGS. 7A and 7B illustrates the unique deformability andcircularity signatures, respectively, for human embryonic stem cells(hESCs) and hESCs undergoing differentiation (day 9). N refers to thenumber of cells represented in the particular scatter plot. Medians forthe two populations are different with statistical significance P<0.001.Unique differences in circularity are also evident (FIG. 7B).

Experiment No. 2—Differentiation of Embryonic Stem Cells

Experiments were also conducted using the system to determine thedeformability of self-renewing mouse embryonic stem cells (mESC)differentiated by adherent (Ad. Diff.) and embryoid body (EB Diff.)methods. Undifferentiated mESC lines were cultured in 5.0% CO₂ at 37° C.on mitomycin C-inactivated CF1 mouse embryonic fibroblast cells. Culturemedium contained KnockOut Dulbecco's modified Eagle's medium (DMEM), 15%fetal calf serum, 1× non-essential amino acids (Invitrogen/GIBCO, 100×concentration), 1× Pen Strep Glutamine (Invitrogen/GIBCO, 100×concentration), 0.055 mM 2-mercaptoethanol (Invitrogen/GIBCO, 1000×concentration, 55 mM), and 55 units leukemia inhibitory factor(Millipore, 106 units/ml). Mouse ESCs were differentiated into eitherembryoid bodies (EBs) as hanging drops or adherent culture on gelatin.Differentiation medium contained KnockOut (DMEM), 15% fetal calf serum,1× nonessential amino acids, 1× Pen Strep Glutamine, and 0.055 mM2-mercaptoethanol. EBs were collected at day 9 for analysis. Foradherent culture, 15,000 cells were plated on gelatin-coated six-wellplates in mESC differentiation medium. Media was changed at day 4, 5 and6. At day 9, cells were collected for analysis.

FIG. 8A illustrates deformability measurements made using the system forundifferentiated mESC, differentiated stem cells with Ad. Diff., anddifferentiated stem cells with EB Diff. FIG. 8B illustrates circularitymeasurements made using the system for undifferentiated mESC,differentiated stem cells with Ad. Diff., and differentiated stem cellswith EB Diff. N refers to the number of cells represented in theparticular scatter plot. Both differentiation methods (Ad. Diff. and EBDiff.) result in an increase in cell stiffness. This statisticallysignificant difference validates findings that differentiation state andassociated cytoskeletal changes may manifest as a difference in cellmechanics. This distinction may find application in identifyingfailed-to-differentiate stem cells destined for implantation which couldotherwise result in tumors in vivo.

Experiment No. 3—Measurement of Metastatic Potential of Cancer Cells

Experiments were also conducted to measure and classify breast cancercells by metastatic potential. FIGS. 9A and 9B illustrate measurementsof deformability and circularity, respectively, for normal cells(MCF10A), cancerous cells (MCF7), and the same cancerous cell linemodified to have increased motility or metastatic potential (modMCF7). Nrefers to the number of cells represented in the particular scatterplot. The MCF10A cell line (ATCC Number: CRL-10317) was cultured inDMEM-F12 with horse serum at a final concentration of 5% (v/v),penicillin/streptomycin 1% (v/v), 20 nM epidermal growth factor, 0.5 μMhydrocortizone, 0.1 μM cholera toxin, and 10 μM insulin. The MCF7 cellline (ATCC Number: HTB-22) was propagated in DMEM-F12 with 0.01 mg/mLbovine insulin and fetal bovine serum at a final concentration of 10%(v/v). Modification to make the cell line more invasive, designated“modMCF7” was carried out by incubation with 400 nM12-Otetradecanoylphorbol-13-acetate (TPA) for 20 hours.

Measurements of both deformability and circularity clearly distinguishnormal cells from cancerous cells and malignant cells. The medians weredetermined by a nonparametric test to be statistically different with aconfidence of P<0.01. This confirms trends observed by other techniquesfor measuring cell mechanical properties with greatly enhancedthroughput, enabling adoption of the technique in settings whereanalysis of diverse cell populations is necessary.

Experiment No. 4—Cytoskeletal Disruption

Experiments were conducted measuring cell deformability of cervicalcarcinoma cells (HeLa) treated with the actin disruptor Latrunculin.HeLa cells were also treated with Nocodazole, a microtubule stabilizer,and measured for cell deformability. The HeLa cell line was maintainedin with DMEM-F12 with 1% (v/v) penicillin/streptomycin and 10% (v/v)fetal bovine serum. To explore the effects of cytoskeleton components ondeformability, microtubules were inhibited with Nocodazole and disruptedactin polymerization with Latrunculin A. Cells were incubated in 0.1 μMLatrunculin A for 4 hours and/or 2 μM nocodazole for 1 hour,respectively, prior to deformability assay.

FIGS. 10A and 10B illustrate deformability and circularity measurements,respectively, made with the system 10 for HeLa cells (control), HeLacells treated with Latrunculin A, and HeLa cells treated withNocodazole. N refers to the number of cells represented in theparticular scatter plot. HeLa cells treated with Latrunculin weremeasured to be significantly more deformable. HeLa cells treated withNocodazole appear more rigid than untreated cells.

Turning now to a particular use of the system 10, the same may be usedto diagnose a disease state. For example, a gating strategy may beemployed wherein large, highly deformable (LDH) cells are used as aproxy for targeting metastatic cancer. For example, the system 10 may beused to test pleural fluid obtained from a subject. The sample may berun through the system 10 to diagnose carcinomas or other malignancies.A threshold may be established requiring a certain percentage of cellsbe LDH; namely the cells have an initial diameter above a certainthreshold value and a deformability above a certain threshold value.FIG. 11A illustrates such a gating strategy whereby the darkened regionwith high deformation and high initial diameter is queried to determinethe overall percentage of cells falling in this region. N refers to thenumber of cells represented in the particular scatter plot. FIG. 11Billustrates a standard negative profile whereby the LHD % is low (inthis case 0.9%). This contrasts with the standard adenocarcinoma profileillustrated in FIG. 11C whereby the LHD % is high (in this case 21.3%).The scatter plots of deformability may also be used in the same way tomake negative determinations. The data can further be used to determinethose instances wherein additional review is needed by a cytologist oradditional testing is needed, atypical cells are present, andinflammation conditions exist. Additional gating schemes other thanstandard thresholding may also be used. For instance, gating functionsestablished via models or prior experimental datasets can be used toidentify a subset of cells based on pre-established criteria based atleast in part on cell size and deformability.

The system 12 may also be used for identifying biomarkers thatcorrespond to various cellular properties. These include, by way ofexample, malignancy, metastatic potential, cell cycle stage,differentiation stage, cytoskeletal integrity, and leukocyte activation.

Advantages of the system 10 over other existing analysis techniquesinclude reduced reagent consumption. Moreover there is less need forlabor given that there is no need for pipetting, centrifugation, etc.The system 10 also allows operation on, smaller volumes without cellloss which might arise in processing steps such as centrifugation andcell handling. The system 10 further has a very high throughput that ismultiple orders of magnitude higher than existing techniques formeasuring cell deformability.

While embodiments have been shown and described, various modificationsmay be made without departing from the scope of the inventive conceptsdisclosed herein. For example, while several embodiments have beendescribed herein it should be appreciated that various aspects orelements are interchangeable with other separately embodiments. Theinvention(s), therefore, should not be limited, except to the followingclaims, and their equivalents.

1-31. (canceled)
 32. A system for measuring a change in a morphologicalparameter of a cell comprising: a substrate containing an inletmicrofluidic channel and one or more outlet microfluidic channels; anextensional region comprising an intersection of the inlet microfluidicchannel and the one or more outlet microfluidic channels, wherein theinlet microfluidic channel and the one or more outlet microfluidicchannels intersect in a T-shaped intersection; a focusing region locatedin the inlet microfluidic channel and upstream of the extensionalregion, wherein the focusing region focuses cells passing there throughinto a stream of cells located along one or more lateral streamlines; anoptical collector configured to capture multiple records of diffractedor refracted light from each cell passing through the extensionalregion; a pump configured to flow a plurality of cells into the inletmicrofluidic channel and through the extensional region, wherein theplurality of cells flow through the extensional region and are nottrapped therein with each cell passing through the extensional region inless than 500 μs; and at least one processor operably coupled to theoptical collector and configured to calculate a change in amorphological parameter of each cell passing through the extensionalregion based on the multiple records, wherein the change in themorphological parameter comprises a dimensional change of each cellpassing through the extensional region.
 33. The system of claim 32,wherein the morphological parameter is selected from the group of cellsize, cell deformability, cell circularity, cell shape, and cellgranularity of cells passing through the extensional region.
 34. Thesystem of claim 32, wherein the one or more outlet microfluidic channelsare arranged substantially perpendicular to the inlet microfluidicchannel.
 35. The system of claim 32, wherein the optical collectorcomprises a photodiode or photomultiplier tube.
 36. The system of claim32, wherein the optical collector comprises a camera.
 37. The system ofclaim 32, wherein the at least one processor is operably connected tothe pump to control a flow rate from the pump.
 38. The system of claim32, wherein the at least one processor is part of a computer.
 39. Amethod of measuring cell deformability comprising: focusing a pluralityof cells in an inlet microfluidic channel dimensioned to carry cellstherein; intersecting the cells focused in the inlet microfluidicchannel with an extensional region formed by one or more outletmicrofluidic channels intersecting with the inlet microfluidic channel,the extensional region configured to apply temporary stress to cellspassing into the extensional region, wherein the cells undergo an abruptchange in velocity in the extensional region; obtaining multiple recordsof diffracted or refracted light from each cell passing through theextensional region; measuring one or more dimensional parameters of thecells from the multiple records; and calculating the deformability of acell using the at least one processor configured to calculate one ormore dimensional parameters occurring during exposure to the stressapplied in the extensional region.
 40. The method of claim 39, whereinthe velocity of the cells in the extensional region abruptly decreasesto substantially zero.
 41. The method of claim 39, wherein the one ormore dimensional parameters comprises measuring the diameter ofindividual cells prior to entering the extensional region.
 42. Themethod of claim 39, wherein the one or more dimensional parameterscomprises measuring maximum and minimum diameters of individual cellsduring exposure to the extensional region.
 43. The method of claim 39,wherein determining deformability comprises calculating a ratio of themaximum cell diameter to the minimum cell diameter.
 44. The method ofclaim 39, further comprising calculating the circularity of a cellwherein circularity is calculated according to the formula 4πA/L²,wherein A comprises cell area and L comprises cell perimeter.
 45. Themethod of claim 39, wherein the inlet microfluidic channel and the oneor more outlet microfluidic channels intersect in a substantiallyorthogonal orientation.
 46. The method of claim 39, further comprisingdisplaying on a display at least one of deformability and circularity asa function of cell diameter calculated by the at least one processor.47. The method of claim 39, further comprising calculating at least oneof membrane elasticity and viscosity.